Perhaps the most critical parameter for successful
PCR is the design of Primers. All things being equal, a poorly designed
primer can result in a PCR reaction that will not work. The primer sequence
determines several things such as the length of the product, its melting
temperature and ultimately the yield. A poorly designed primer can result
in little or no product due to non-specific amplification and/or primer-dimer
formation, which can become competitive enough to suppress product formation.
This application note is provided to give rules that should be taken into
account when designing primers for PCR. More comprehensive coverage of this
subject can be found elswhere(1).

Primer selection

Several variables must be taken into account when designing PCR Primers.
Among the most critical are:

Since both specificity and the temperature and time of annealing are
at least partly dependent on primer length, this parameter is critical
for successful PCR. In general, oligonucleotides between 18 and 24 bases
are extremely sequence specific, provided that the annealing temperature
is optimal. Primer length is also proportional to
annealing efficiency:
in general, the longer the primer, the more inefficient the annealing.
With fewer templates primed at each step, this can result in a significant
decrease in amplified product. The primers should not be too short, however,
unless the application specifically calls for it. As discussed below,
the goal should be to design a primer with an annealing temperature of
at least 50C.

The relationship between annealing temperature and melting temperature
is one of the Black Boxes of PCR. A general rule-of-thumb
is to use an annealing temperature that is 5C lower than the melting
temperature. Thus, when aiming for an annealing temperature of at least
50C, this corresponds to a primer with a calculated melting temperature(Tm)
~55C. Often, the annealing temperature determined in this fashion
will not be optimal and empirical experiments will have to be performed
to determine the optimal temperature. This is most easily accomplished
using a gradient thermal cycler like Eppendorf's Mastercycler gradient.

Melting Temperature (Tm)

It is important to keep in mind that there are two primers added to a
PCR reaction. Both of the oligonucleotide primers should be designed such
that they have similar melting temperatures. If primers are mismatched
in terms of Tm, amplification will be less efficient or may not work at
all since the primer with the higher Tm will mis-prime at lower temperatures
and the primer with the lower Tm may not work at higher temperatures.

The melting temperatures of oligos are most accurately calculated using
nearest neighbor thermodynamic calculations with the formula:

Tmprimer =
?H [?S+ R ln (c/4)] 273.15C + 16.6 log 10 [K+]

where H is the enthalpy and S is the entropy for helix formation, R is
the molar gas constant and c is the concentration of primer. This is most
easily accomplished using any of a number of primer design software packages
on the market(3). Fortunately, a good working approximation of this value
(generally valid for oligos in the 1824 base range) can be calculated
using the formula:

Tm = 2(A+T) + 4(G+C).

The table below shows calculated values for primers of various lengths
using this equation, which is known as the Wallace formula, and assuming
a 50% GC content(4).

The temperatures calculated using Wallace's
rule are inaccurate at the extremes of this chart.

In addition to calculating the melting temperatures of the primers, care
must be taken to ensure that the melting temperature of the product is
low enough to ensure 100% melting at 92C. This parameter will help
ensure a more efficient PCR, but is not always necessary for successful
PCR. In general, products between 100600 base pairs are efficiently
amplified in many PCR reactions. If there is doubt, the product Tm can
be calculated using the formula:

Tm = 81.5 + 16.6 (log10[K+] + 0.41 (%G+C)675/length.

Under standard PCR conditions of 50 mM KCL, this reduces to(3):

Tm = 59.9 + 0.41 (%G+C) 675/length

Specificity

As mentioned above, primer specificity is at least partly dependent on
primer length. It is evident that there are many more unique 24 base oligos
than there are 15 base pair oligos. That bein
g said, primers must be chosen
so that they have a unique sequence within the template DNA that is to
be amplified. A primer designed with a highly repetitive sequence will
result in a smear when amplifying genomic DNA. However, the same primer
may give a single band if a single clone from a genomic library is amplified.

Because Taq Polymerase is active over a broad range of temperatures,
primer extension will occur at the lower temperatures of annealing. If
the temperature is too low, non-specific priming may occur which can be
extended by the polymerase if there is a short homology at the 3' end.
In general, a melting temperature of 55C 72C gives the
best results (Note that this corresponds to a primer length of 1824
bases using Wallace's rule above).

Complementary Primer Sequences

Primers need to be designed with absolutely no intra-primer homology
beyond 3 base pairs. If a primer has such a region of self-homology, snap
back, partially double-stranded structures, can occur which will
interfere with annealing to the template.

Another related danger is inter-primer homology. Partial homology in
the middle regions of two primers can interfere with hybridization. If
the homology should occur at the 3' end of either primer, Primer dimer
formation will occur which, more often than not, will prevent the formation
of the desired product via competition.

G/C content and Polypyrimidine (T, C) or
polypurine (A, G) stretches

The base composition of primers should be between 45% and 55% GC. The
primer sequence must be chosen such that there is no PolyG or PolyC stretches
tha
t can promote non-specific annealing. Poly A and Poly T stretches are
also to be avoided as these will breath and open up stretches
of the primer-template complex. This can lower the efficiency of amplification.
Polypyrimidine (T, C) and polypurine (A, G) stretches should also be avoided.
Ideally the primer will have a near random mix of nucleotides, a 50% GC
content and be ~20 bases long. This will put the Tm in the range of 56C
62C(1).

3-end Sequence

It is well established that the 3' terminal position in PCR primers is
essential for the control of mis-priming(5). We have already explored
the problem of primer homologies occurring at these regions. Another variable
to look at is the inclusion of a G or C residue at the 3' end of primers.
This GC Clamp helps to ensure correct binding at the 3' end
due to the stronger hydrogen bonding of G/C residues. It also helps to
improve the efficiency of the reaction by minimizing any breathing
that might occur.

Conclusion

It is essential that care is taken in the design of primers for PCR.
Several parameters including the length of the primer, %GC content and
the 3' sequence need to be optimized for successful PCR. Certain of these
parameters can be easily manually optimized while others are best done
with commercial computer programs. In any event, careful observance of
the general rules of primer design will help ensure successful experiments.

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